Cytokines are key mediators of innate and adaptive immunity. Many cytokines have been used for therapeutic purposes in patients, such as those with advanced cancer, but their administration is typically associated with severe toxicity, hampering dose escalation to therapeutically active regimens and their development as anticancer drugs, for example. To overcome these problems, the use of Immunocytokines' (i.e. cytokines fused to antibodies or antibody fragments) has been proposed, with the aim to concentrate the immune-system stimulating activity at the site of disease while sparing normal tissues (Savage et al., 1993; Schrama et al., 2006; Neri et al. 2005; Dela Cruz et al., 2004; Reisfeld et al., 1997; Konterman et al., 2012).
For example, several pro-inflammatory immunocytokines (e.g., those based on IL2, IL12, IL15, TNF) have been shown to display a potent anti-tumoural effect in mouse models of cancer (Borsi et al. 2003; Carnemolla et al., 2002; Frey et al., 2010; Kaspar et al., 2007; Pasche et al., 2012). In contrast, anti-inflammatory immunocytokines (e.g., those based on IL10) have been shown to be capable of conferring a therapeutic benefit in mouse models of chronic inflammatory conditions (rheumatoid arthritis, endometriosis [Schwager et al. 2011; Schwager et al., 2009]) but have no impact on tumour growth.
Antibodies specific to splice-isoforms of fibronectin and of tenascin-C have been described as vehicles for pharmacodelivery applications, as these antigens are virtually undetectable in the normal healthy adult (with the exception of the placenta, endometrium and some vessels in the ovaries) while being strongly expressed in the majority of solid tumours and lymphomas (Brack et al., 2006; Pedretti et al, 2009; Schliemann et al. 2009). For example, antibodies F8 and L19, specific to the alternatively-spliced EDA and EDB domains of fibronectin, respectively, and anti-tenascin C antibody F16 (Brack et al., 2006; Villa et al., 2008; Viti et al., 1999), have been employed for the development of armed antibodies, some of which have begun clinical testing in oncology and in rheumatology (Eigentler et al., 2011; Papadia et al., 2012). The tumour targeting properties of these antibodies have also been documented in mouse models of cancer and in patients.
Interleukin 22 (IL22) is a 17 kDa globular cytokine belonging to the IL-10 family, which is mainly secreted by NK cells, dendritic cells and T-cells (Murphy 2012). It contains two intramolecular disulfide bonds and three N-linked glycosylation sites. Biological functions of IL22 include involvement in tissue protection, autoimmunity and inflammation. Secreted by lamina propria effector T-cells in the intestine, it induces mucin production, antimicrobial, proliferative and antiapoptotic pathways, which prevent tissue damage and promote epithelial repair. (Li et al., 2014). We investigated whether IL22 could be successfully fused to a vascular targeting antibody.
Cytokines can be conjugated to antibody molecules to produce immunocytokines as mentioned above. However, not all immunocytokines retain, for example, the in vivo targeting properties of the parental antibody (Pasche & Neri, 2012) or expected activities. The preparation of immunocytokines with therapeutic effects, such as anti-inflammatory activity, is therefore far from straightforward.
The preparation of conjugates comprising a mouse IgG1 Fc fused to the N-terminus or C-terminus of mouse IL-22 is described in Smith et al. (2013). These conjugates were prepared with a view to providing a more potent and longer-lasting IL-22R agonist compared with rIL-22. The purpose of the Fc region in this instance was therefore not to target IL22 to regions of disease, as was the case with the immunocytokines described in the preceding paragraph. In addition, Smith et al. (2013) shows that when the C-terminus of a murine Fc portion was fused to IL22 (Fc-IL-22), the fusion protein had higher activity than recombinant IL22, while when the N-terminus of a murine Fc portion was fused to IL22 (IL-22-Fc), the fusion protein had only minimal activity in vitro and no detectable activity in vivo.